Pre-exposure prophylaxis (PrEP) is a new and effective HIV-1 prevention strategy. However, individuals who become HIV-1 infected despite PrEP will have weeks or months of PrEP exposure during acute infection before seroconversion is detected, potentially promoting the selection of drug-resistant HIV-1. Preliminary data suggest that resistance may be very rare in PrEP breakthrough infections. However, these data are largely reliant on traditional consensus genotyping, which is unable to detect resistance present at low frequencies of the viral population. As we have learned from studies of prophylaxis to prevent mother-to-child transmission of HIV-1, resistance that arises from antiretrovirals used for prevention require more sensitive methods than traditional consensus genotyping, because minority variants (i.e., comprising <20% of viruses in an individual) can persist after drug exposure and contribute to subsequent failure of antiretroviral therapy. Therefore, studies that utilize high-sensitivity resistance testing are needed to rule out that low-frequency (minority) resistant variants may develop in individuals infected with HIV-1 while taking PrEP. Most studies of PrEP resistance have been conducted in the context of carefully conducted clinical trials. Here we propose to assess low-frequency antiretroviral resistance in cohorts of African heterosexuals receiving PrEP in both: 1) the Partners PrEP clinical trial along with its follow-up cohort, in which PrEP adherence has been high and 2) an NIH-funded PrEP demonstration project initiating in 2012 that will implement PrEP in a real-world setting. The Partners PrEP Study was a randomized placebo-controlled trial of daily tenofovir (TDF) and emtricitabine/tenofovir (FTC/TDF) among HIV-1 uninfected partners in 4758 African heterosexual HIV-1 serodiscordant couples, and is the largest oral PrEP trial to date. In the Partners PrEP trial, oral TDF and FTC/TDF reduced HIV-1 acquisition by 67% (p<0.001) and 75% (p<0.001), respectively. Based on the efficacy of Partners PrEP, a demonstration project is being conducted to evaluate strategies for PrEP implementation in high-risk HIV-1 serodiscordant couples. We will determine the occurrence of low-frequency resistance in seroconverters in these cohorts using a highly sensitive ultra-deep sequencing assay. We hypothesize that previous studies have underestimated the resistance that emerges in seroconverters taking PrEP. We believe that resistance will be more accurately assessed using highly sensitive assays in cohorts which reflect PrEP implementation programs, where counseling and monitoring is less frequent than in a clinical trial setting. These studies will address a critical gap in understanding how commonly resistance, including low-frequency resistant variants, is selected for in HIV-1 seroconverters receiving PrEP. Understanding the risk of developing resistance from PrEP and the potential mechanisms that lead to resistance in this context is crucial before this prevention strategy is implemented on a larger scale.